In diabetes, the accumulation of sorbitol is believed to be a key factor in initiating cataract, retinopathy, and neuropathy. We are interested in controlling the accumulation of sorbitol by regulating the action of two enzymes of the sorbitol pathway: Aldose reductase (AR) reduces glucose to sorbitol, and sorbitol dehydrogenase (SDH) oxidizes sorbitol to fructose. Our aim is to design innovative methods to inhibit the action of AR or increase SDH with the purpose of reducing sorbitol accumulation in diabetic tissues. Site-directed mutagenesis of AR has been a major priority of our laboratory. We have made amino acid substitutions in the rat and human AR and determined that some of these changes affect the kinetics of the protein with its substrate. For example, when cysteine at position 298 is changed to a serine, the kinetics of glyceraldehyde increases 2-fold, while xylose decreases to 15% of normal. These structure/function studies should help define the active site and locate the target areas of the current AR inhibitors. Another strategy to control sorbitol accumulation is to regulate SDH. We have initiated studies to understand the structure of this protein and its action in vitro. The sequence of human SDH has been determined using cDNA analysis. Northern blots indicate that levels of SDH mRNA are higher in kidney than in other tissues, such as lung, liver, brain, and heart. A major portion of the SDH gene also has been obtained. These tools will allow us to study the regulation of SDH at the gene level.